Methylotuvimicrobium Alcaliphilum 20Z Research Articles

Methylotuvimicrobium alcaliphilum 20Z based ectoine production from various industrial CH4 sources: Cultivation optimization and comprehensive analysis

Methanotroph-based CH4 conversion technology is being widely studied due to its advantages of mild operating conditions and environmental friendliness. In this study, operating strategies were investigated for industrial CH4 sources in order to widely disseminate methanotroph-based CH4 conversion technology using the promising strain, Methylotuvimicrobium alcaliphilum 20Z. The optimal O2/CH4 ratio for extremely different CH4 concentrations was explored based on kinetic-based simulation for biological reaction, and experimentally proven using fermenter-scale experiments. As a result, when high CH4 concentration is used, ectoine production rate can be maximized (33.8 mg/L·h) at an O2/CH4 ratio of 1 while favoring the “production of metabolites”. On the other hand, in the low CH4 concentration case, when changing from the general O2/CH4 ratio of 4 to the optimal O2/CH4 ratio (i.e., 1), although the improvement in ectoine concentration was not noticeable (3.9 to 7.5 mg/L·h), the CH4 conversion rate rapidly decreased (50 to 20 %), suggesting that the focus in the low concentration case should be on “removal of CH4”. In the case of biogas, the rapid pH drop caused by the large amount of CO2 in the feed is offset by a buffer solution, and the concentration of Na+ ions increases during the overall reaction time. Therefore, the production of metaboiltes can be maximized while simultaneously controlling pH and Na+ ion through continuous operation with a high dilution rate (19.8 mg/L·h). Furthermore, major performance indicators for each industrial gas were derived through comprehensive comparative analysis, through which the “dual-functions” of methanotrophs could be emphasized. This study provides a practical and strategic approach for methanotrophs that can flexibly respond to diverse industrial CH4 sources.

Methane capture, utilization, and sequestration technology based on biological ectoine production using Methylotuvimicrobium alcaliphilum 20Z

Methanotroph-based CH4 conversion technology is a promising alternative to conventional CH4 utilization methods that rely on energy-intensive CO2 capture processes. In this study, we introduce a novel CH4 capture, utilization, and sequestration (MCUS) technology as a proof-of-concept for eco-friendly CH4 conversion that is applicable to various methanotroph strains. We validated this hypothesis by evaluating ectoine production in Methylotuvimicrobium alcaliphilum 20Z. Cultivation experiments were conducted using pure O2 pulse injection to facilitate the separation of the CO2 generated during CH4 oxidation by concentrating it. To verify the MCUS concept systematically, we performed a model-based process analysis. The operational strategies for the reactors were developed using experiment-based kinetics and reactor models, followed by the design of a rigorous process flow diagram. Economic and environmental impact analyses were conducted to compare MCUS with air-based cultures and existing oxidative CH4 conversion technologies. The key advantage of MCUS is its significantly lower CO2 emissions (0.71 kg-CO2 eq/kg-CH4) than oxidative CH4 utilization (>2.75 kg-CO2 eq/kg-CH4). Moreover, the net present value per CH4 molecule was estimated at 0.7 $/kg-CH4, substantially higher than existing oxidative conversion methods (0.03 $/kg-CH4). Additionally, compared with air-based cultivation, the MCUS process exhibits superior economic and environmental performances. We used methanotrophs for CH4 “capture” and “utilization,” effectively leveraging “sequestration” to convert emissions into pure CO2. The proposed MCUS technology holds promise for practical applications of methanotrophs in producing various high-value-added products, emphasizing eco-friendly approaches.

Interchangeability of class I and II fumarases in an obligate methanotroph Methylotuvimicrobium alcaliphilum 20Z.

The methanotrophic bacterium Methylotuvimicrobium alcaliphilum 20Z is an industrially promising candidate for bioconversion of methane into value-added chemicals. Here, we have study the metabolic consequences of the breaking in the tricarboxylic acid (TCA) cycle by fumarase knockout. Two fumarases belonging to non-homologous class I and II fumarases were obtained from the bacterium by heterologous expression in Escherichia coli. Class I fumarase (FumI) is a homodimeric enzyme catalyzing the reversible hydration of fumarate and mesaconate with activities of ~94 and ~81 U mg-1 protein, respectively. The enzyme exhibited high activity under aerobic conditions, which is a non-typical property for class I fumarases characterized to date. The calculation of kcat/S0.5 showed that the enzyme works effectively with either fumarate or mesaconate, but it is almost four times less specific to malate. Class II fumarase (FumC) has a tetrameric structure and equal activities of both fumarate hydration and malate dehydration (~45 U mg-1 protein). Using mutational analysis, it was shown that both forms of the enzyme are functionally interchangeable. The triple mutant strain 20Z-3E (ΔfumIΔfumCΔmae) deficient in the genes encoding the both fumarases and the malic enzyme accumulated 2.6 and 1.1 mmol g-1 DCW fumarate in the medium when growing on methane and methanol, respectively. Our data suggest the redundancy of the metabolic node in the TCA cycle making methanotroph attractive targets for modification, including generation of strains producing the valuable metabolites.

Development of a novel methanotrophic platform to produce ectoine from methane and lignocellulose-derived sugars

Methane is considered a promising carbon source in the field of biotechnology. Using methane as the carbon source for producing chemicals reduces manufacturing costs and tackles the issue of global warming. Like methane, lignocellulose biomass is also an inexpensive and ample carbon source, which could be used for industrial biomanufacturing. In this study, Methylotuvimicrobium alcaliphilum 20Z was genetically modified to simultaneously consume methane and lignocellulose-derived sugars (glucose and xylose) to produce ectoine, a native osmoprotectant. The xylose utilization pathway, comprising xylA (xylose isomerase) and xylB (xylulokinase) genes from Escherichia coli, was first integrated into the chromosome of M. alcaliphilum 20Z. Further expression of the glucose utilization, consisting of glf (glucose facilitator) from Zymmonas mobilis, glk (endogenous glucokinase), and pgi (glucose 6-phosphate isomerase) from E. coli, made the engineered strain 20ZXG consume methane, xylose, and glucose concurrently. Cultivation on three substrates upregulated the Embden–Meyerhof–Parnas and ectoine biosynthesis pathways of 20Z. As a result, the ectoine content produced by 20ZXG grown on the three substrates was 1.7-fold enhanced compared with that obtained on methane solely. Furthermore, the knock-out of ectoine biosynthesis repressor (ectR1) led to an output of 37.93 ± 3.27 mg/g-DCW, which was the highest quantity of ectoine produced after 48 h of cultivation in the presence of three substrates and was 2.3-fold higher than the initial output.

Insights into C1 and C3 assimilation pathways in type I methanotrophic bacterium from co-production of 1,2-propanediol and lactate

Methanotrophic bacteria are attractive hosts for mining metabolic pathways of C1 assimilation to produce value-added products. Herein, the type I methanotroph Methylotuvimicrobium alcaliphilum 20Z was employed to explore the carbon flux from methane and methanol via the EMP pathway to produce 1,2-propanediol (1,2-PDO). The production of 1,2-PDO on methane was found to be mainly restricted by the lower carbon flux toward the EMP pathway. The co-utilization of C1 substrates and glycerol (C3) could contribute to enhance 1,2-PDO. Lactate was co-produced in much higher amounts than 1,2-PDO. This unexpected product was probably derived from lactaldehyde by inherent aldehyde dehydrogenases. The 1,2-PDO production without increased accumulation of lactate was observed via establishing the acetol–based pathway by propane utilization with the overexpression of pmoD. This is the first study to provide experimental insights into the operation of metabolic routes for 1,2-PDO and lactate co-production from C1 and C3 compounds in methanotrophs.

Engineering type I methanotrophic bacteria as novel platform for sustainable production of 3-hydroxybutyrate and biodegradable polyhydroxybutyrate from methane and xylose

Methylotuvimicrobium alcaliphilum20Z recombinant strain co-utilizing methane and xylose from anthropogenic activities and lignocellulose biomassis a promising cell factory platform. In this study, the production of (R)-3-hydroxybutyrate and poly (3-hydroxybutyrate) inM. alcaliphilum20Z was demonstrated. The production of (R)-3-hydroxybutyrate was optimized by introducing additional thioesterase, and a tunable genetic module. The final recombinant strain produced the highest titer of 334.52 ± 2 mg/L (R)-3-hydroxybutyrate (yield of 1,853 ± 429 mg/g dry cell weight). The poly (3-hydroxybutyrate) yielded 1.29 ± 0.08% (w/w) from methane and xylose in one-stage cultivation. Moreover, the study demonstrated the importance of pathway reversibility as an effective design strategy for balancing the driving force and intermediate accumulation. This is the first demonstration of the production ofbiodegradablepoly (3-hydroxybutyrate) from methane in type I methanotrophs, which is a key step toward sustainable biomanufacturing and carbon–neutral society.

Biological production of 2-propanol from propane using a metabolically engineered type I methanotrophic bacterium

2-Propanol is a widely used industrial solvents. Herein, we employed a unique feature of type I methanotrophic bacterium Methylotuvimicrobium alcaliphilum 20Z possessing only particulate methane monooxygenase (pMMO) for one-step direct production of pure 2-propanol from propane. By maintaining cell growth on glycerol, and after deletion of both Ca2+-dependent and La3+-dependent methanol dehydrogenases, propane was converted to 2-propanol by pMMO. Although most of the 2-propanol produced was further oxidized to acetone, deletion of active alcohol dehydrogenase, concomitant with synchronous overexpression of secondary alcohol dehydrogenase, significantly inhibited such undesirable oxidation. As a result, a remarkable enhancement (263 mg/L) of 2-propanol was achieved for 120 h by increasing cell growth with a supply of 50% (v/v) propane in headspace. This is the first demonstration to develop an engineered methanotrophic strain for the one-step direct production of pure 2-propanol from propane using one-phase cultivation without the supply of chemical inhibitors or additional reducing-power sources.

Development of an engineered methanotroph-based microbial platform for biocatalytic conversion of methane to phytohormone for sustainable agriculture

Methane is an important target for reducing greenhouse gases emission, owing to its abundance and highly global warming potential. Bioconversion of methane to value-added products, therefore, has become a promising and sustainable approach for industrial biomanufacturing. Herein, we metabolically engineered an obligate methanotroph Methylotuvimicrobium alcaliphilum 20Z to overproduce L-tryptophan and its derivative indole 3-acetic acid (IAA), a phytohormone, from methane. The engineered RS00 strain initially produced tryptophan from methane with a titer of 39.6 mg/L at 144 h of cultivation. The highest titer of IAA of 10.5 mg/L from methane was obtained in RS01 strain under Ptac promoter. Moreover, xylose, a carbon source derived from lignocellulosic biomass used as co-substrate, could enhance the titers of tryptophan and IAA in RS00 and RS01 strains by 60% and 82%, respectively. Remarkably, we proposed a proof-of-concept for the development of methanotroph-based plant growth-promoting bacteria (PGPB) which can utilize and convert methane into phytohormones to induce beneficial adaptation and growth promotion of plants. In this study, under saline-alkaline conditions, the germination percentage and elongation of the shoots and roots of wheat seeds treated with the IAA-producing strain were significantly improved, being 114%, 199% and 362% higher than those of control treatments, respectively. Our findings provide a novel methanotrophic platform for the conversion of renewable sources to value-added products. Additionally, the strategies and concepts presented here could be further expanded and applied to other methanotrophic hosts to develop biofertilizers that promote plant growth while mitigating methane and reducing chemical fertilizer use for sustainable agriculture production.

Enhancing Sesquiterpenoid Production from Methane via Synergy of the Methylerythritol Phosphate Pathway and a Short-Cut Route to 1-Deoxy-D-xylulose 5-Phosphate in Methanotrophic Bacteria.

Sesquiterpenoids are one of the most diverse classes of isoprenoids which exhibit numerous potentials in industrial biotechnology. The methanotrophs-based methane bioconversion is a promising approach for sustainable production of chemicals and fuels from methane. With intrinsic high carbon flux though the ribulose monophosphate cycle in Methylotuvimicrobium alcaliphilum 20Z, we demonstrated here that employing a short-cut route from ribulose 5-phosphate to 1-deoxy-d-xylulose 5-phosphate (DXP) could enable a more efficient isoprenoid production via the methylerythritol 4-phosphate (MEP) pathway, using α-humulene as a model compound. An additional 2.8-fold increase in α-humulene production yield was achieved by the fusion of the nDXP enzyme and DXP reductase. Additionally, we utilized these engineering strategies for the production of another sesquiterpenoid, α-bisabolene. The synergy of the nDXP and MEP pathways improved the α-bisabolene titer up to 12.24 ± 0.43 mg/gDCW, twofold greater than that of the initial strain. This study expanded the suite of sesquiterpenoids that can be produced from methane and demonstrated the synergistic uses of the nDXP and MEP pathways for improving sesquiterpenoid production in methanotrophic bacteria.

Enzymes of an alternative pathway of glucose metabolism in obligate methanotrophs

Aerobic methanotrophic bacteria utilize methane as a growth substrate but are unable to grow on any sugars. In this study we have shown that two obligate methanotrophs, Methylotuvimicrobium alcaliphilum 20Z and Methylobacter luteus IMV-B-3098, possess functional glucose dehydrogenase (GDH) and gluconate kinase (GntK). The recombinant GDHs from both methanotrophs were homotetrameric and strongly specific for glucose preferring NAD+ over NADP+. GDH from Mtm. alcaliphilum was most active at pH 10 (Vmax = 95 U/mg protein) and demonstrated very high Km for glucose (91.8 ± 3.8 mM). GDH from Mb. luteus was most active at pH 8.5 (Vmax = 43 U/mg protein) and had lower Km for glucose (16 ± 0.6 mM). The cells of two Mtm. alcaliphilum double mutants with deletions either of the genes encoding GDH and glucokinase (gdh─/glk─) or of the genes encoding gluconate kinase and glucokinase (gntk─/glk─) had the lower glycogen level and the higher contents of intracellular glucose and trehalose compared to the wild type strain. The gntk─/glk─ knockout mutant additionally accumulated gluconic acid. These data, along with bioinformatics analysis, demonstrate that glycogen derived free glucose can enter the Entner–Doudoroff pathway or the pentose phosphate cycle in methanotrophs, bypassing glycolysis via the gluconate shunt.

Sustainable biosynthesis of chemicals from methane and glycerol via reconstruction of multi-carbon utilizing pathway in obligate methanotrophic bacteria.

SummaryObligate methanotrophic bacteria can utilize methane, an inexpensive carbon feedstock, as a sole energy and carbon substrate, thus are considered as the only nature‐provided biocatalyst for sustainable biomanufacturing of fuels and chemicals from methane. To address the limitation of native C1 metabolism of obligate type I methanotrophs, we proposed a novel platform strain that can utilize methane and multi‐carbon substrates, such as glycerol, simultaneously to boost growth rates and chemical production in Methylotuvimicrobium alcaliphilum 20Z. To demonstrate the uses of this concept, we reconstructed a 2,3‐butanediol biosynthetic pathway and achieved a fourfold higher titer of 2,3‐butanediol production by co‐utilizing methane and glycerol compared with that of methanotrophic growth. In addition, we reported the creation of a methanotrophic biocatalyst for one‐step bioconversion of methane to methanol in which glycerol was used for cell growth, and methane was mainly used for methanol production. After the deletion of genes encoding methanol dehydrogenase (MDH), 11.6 mM methanol was obtained after 72 h using living cells in the absence of any chemical inhibitors of MDH and exogenous NADH source. A further improvement of this bioconversion was attained by using resting cells with a significantly increased titre of 76 mM methanol after 3.5 h with the supply of 40 mM formate. The work presented here provides a novel framework for a variety of approaches in methane‐based biomanufacturing.

Malyl-CoA lyase provides glycine/glyoxylate synthesis in type I methanotrophs.

The biochemical routes for assimilation of one-carbon compounds in bacteria require many clarifications. In this study, the role of malyl-CoA lyase in the metabolism of the aerobic type I methanotroph Methylotuvimicrobium alcaliphilum 20Z has been investigated by gene inactivation and biochemical studies. The functionality of the enzyme has been confirmed by heterologous expression in Escherichia coli. The mutant strain lacking Mcl activity demonstrated the phenotype of glycine auxotrophy. The genes encoding malyl-CoA lyase are present in the genomes of all methanotrophs, except for representatives of the phylum Verrucomicrobium. We suppose that malyl-CoA lyase is the enzyme that provides glyoxylate and glycine synthesis in the type I methanotrophs supporting carbon assimilation via the serine cycle in addition to the major ribulose monophosphate cycle.

Methanotrophic microbial cell factory platform for simultaneous conversion of methane and xylose to value-added chemicals

• Methane and xylose are promising feedstock due to their abundance and low cost. • A novel cell factory platform for co-conversion of methane and xylose was developed. • Engineered methanotroph produced value-added shinorine from methane and xylose. • Biosynthesis of 2,3-BDO, acetoin, and 3-hydroxybutyric acid were also demonstrated. • This work provides a promising cell factory platform for sustainable manufacturing. Biotechnological production of value-added products from sustainable resources such as C1 gas and lignocellulosic biomass is a practical solution for sustainable manufacturing. Methane and xylose derived from biogas and biomass can be promising carbon feedstock for chemical biomanufacturing due to their abundance and low cost. Herein, we developed a novel cell factory platform for highly efficient biosynthesis of chemicals from methane and xylose. By taking advantage of intrinsic high carbon flux through non-oxidative part of pentose phosphate pathway in ribulose monophosphate (RuMP) cycle of Methylotuvimicrobium alcaliphilum 20Z, we have metabolically engineered M. alcaliphilum 20Z to co-utilize methane and xylose as carbon and energy sources along with synthesizing of non-oxidative RuMP cycle-derived secondary metabolite, shinorine - a sunscreen material. Importantly, the final titer and yield of shinorine using the engineered methanotrophic biocatalyst were comparable to those from industrial workhorses. In order to confirm the versatility of this concept, the biocatalyst was applied for efficient biosynthesis of other bioproducts including 2,3-butanediol, acetoin, and 3-hydroxybutyric acid from methane and xylose. This work provides a novel and promising methanotrophic cell factory platform for chemical production from abundant methane and renewable xylose in a sustainable way.

Construction of a Type-I Metanotroph with Reduced Capacity for Glycogen and Sucrose Accumulation

Mutant strains of the halotolerant type-I methanotroph Methylotuvimicrobium alcaliphilum 20Z with an inactivated sps gene, which encodes sucrose phosphate synthase, and the deletion of a gene cluster for the synthesis and degradation of glycogen were obtained. Blockage of the synthesis of sucrose and glycogen increased the protein content in cells but slightly reduced the growth rate of the methanotroph grown on methane. It is shown that relatively stable methanotroph growth is possible without synthesis of the storage carbon compounds.

Metabolic role of pyrophosphate-linked phosphofructokinase pfk for C1 assimilation in Methylotuvimicrobium alcaliphilum 20Z

BackgroundMethanotrophs is a promising biocatalyst in biotechnological applications with their ability to utilize single carbon (C1) feedstock to produce high-value compounds. Understanding the behavior of biological networks of methanotrophic bacteria in different parameters is vital to systems biology and metabolic engineering. Interestingly, methanotrophic bacteria possess the pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) instead of the ATP-dependent 6-phosphofructokinase, indicating their potentials to serve as promising model for investigation the role of inorganic pyrophosphate (PPi) and PPi-dependent glycolysis in bacteria. Gene knockout experiments along with global-omics approaches can be used for studying gene functions as well as unraveling regulatory networks that rely on the gene product.ResultsIn this study, we performed gene knockout and RNA-seq experiments in Methylotuvimicrobium alcaliphilum 20Z to investigate the functional roles of PPi-PFK in C1 metabolism when cells were grown on methane and methanol, highlighting its metabolic importance in C1 assimilation in M. alcaliphilum 20Z. We further conducted adaptive laboratory evolution (ALE) to investigate regulatory architecture in pfk knockout strain. Whole-genome resequencing and RNA-seq approaches were performed to characterize the genetic and metabolic responses of adaptation to pfk knockout. A number of mutations, as well as gene expression profiles, were identified in pfk ALE strain to overcome insufficient C1 assimilation pathway which limits the growth in the unevolved strain.ConclusionsThis study first revealed the regulatory roles of PPi-PFK on C1 metabolism and then provided novel insights into mechanism of adaptation to the loss of this major metabolic enzyme as well as an improved basis for future strain design in type I methanotrophs.

Unlocking the biosynthesis of sesquiterpenoids from methane via the methylerythritol phosphate pathway in methanotrophic bacteria, using α-humulene as a model compound

Isoprenoids are an abundant and diverse class of natural products with various applications in the pharmaceutical, cosmetics and biofuel industries. A methanotroph-based biorefinery is an attractive scenario for the production of a variety of value-added compounds from methane, because methane is a promising alternative feedstock for industrial biomanufacturing. In this study, we metabolically engineered Methylotuvimicrobium alcaliphilum 20Z for de novo synthesis of a sesquiterpenoid from methane, using α-humulene as a model compound, via optimization of the native methylerythritol phosphate (MEP) pathway. Expression of codon-optimized α-humulene synthase from Zingiber zerumbet in M. alcaliphilum 20Z resulted in an initial yield of 0.04 mg/g dry cell weight. Overexpressing key enzymes (IspA, IspG, and Dxs) for debottlenecking of the MEP pathway increased α-humulene production 5.2-fold compared with the initial strain. Subsequently, redirecting the carbon flux through the Embden–Meyerhof–Parnas pathway resulted in an additional 3-fold increase in α-humulene production. Additionally, a genome-scale model using flux scanning based on enforced objective flux method was used to identify potential overexpression targets to increase flux towards isoprenoid production. Several target reactions from cofactor synthesis pathways were probed and evaluated for their effects on α-humulene synthesis, resulting in α-humulene yield up to 0.75 mg/g DCW with 18.8-fold enhancement from initial yield. This study first demonstrates production of a sesquiterpenoid from methane using methanotrophs as the biocatalyst and proposes potential strategies to enhance production of sesquiterpenoid and related isoprenoid products in engineered methanotrophic bacteria.

Ectoine degradation pathway in halotolerant methylotrophs.

BackgroundMicroorganisms living in saline environments are forced to regulate turgor via the synthesis of organic osmoprotective compounds. Microbial adaptation to fluctuations in external salinity includes degradation of compatible solutes. Here we have examined the biochemical pathway of degradation of the cyclic imino acid ectoine, the major osmoprotector in halotolerant methane-utilizing bacteria.MethodsThe BLAST search of the genes involved in ectoine degradation in the halotolerant methanotroph Methylotuvimicrobium alcaliphilum 20Z was performed with the reference sequences of Halomonas elongata. The genes for the key enzymes of the pathway were disrupted by insertion mutagenesis and the cellular metabolites in the methanol extracts of mutant cells were analyzed by HPLC. The doeA gene from Mm. alcaliphilum 20Z was heterologously expressed in Escherichia coli to identify the product of ectoine hydrolysis catalyzed by ectoine hydrolase DoeA.ResultsWe have shown that the halotolerant methanotroph Mm. alcaliphilum 20Z possesses the doeBDAC gene cluster coding for putative ectoine hydrolase (DoeA), Nα-acetyl-L-2,4-diaminobutyrate deacetylase (DoeB), diaminobutyrate transaminase (DoeD) and aspartate-semialdehyde dehydrogenase (DoeC). The deletion of the doeA gene resulted in accumulation of the higher level of ectoine compared to the wild type strain. Nγ-acetyl-L-2,4-diaminobutyrate (Nγ-acetyl-DAB), a substrate for ectoine synthase, was found in the cytoplasm of the wild type strain. Nα-acetyl-L-2,4-diaminobutyrate (Nα-acetyl-DAB), a substrate for the DoeB enzyme, appeared in the cells as a result of exposure of the doeB mutant to low osmotic pressure. The genes for the enzymes involved in ectoine degradation were found in all aerobic methylotrophs capable of ectoine biosynthesis. These results provide the first evidence for the in vivo operation of the ectoine degradation pathway in methanotrophs and thus expand our understanding of the regulation mechanisms of bacterial osmoadaptation.ConclusionsDuring adaptation to the changes in external osmolarity, halophilic and halotolerant methylotrophs cleave ectoine, thereby entering the carbon and nitrogen of the compatible solute to the central metabolic pathways. The biochemical route of ectoine degradation in the halotolerant methanotroph Mm. alcaliphilum 20Z is similar to that in heterotrophic halophiles. We have shown that ectoine hydrolase DoeA in this methanotroph hydrolyzes ectoine with the formation of the only isomer: Nα-acetyl-DAB. All aerobic methylotrophs capable of ectoine biosynthesis harbor the genetic determinants for ectoine degradation.

PheS AG Based Rapid and Efficient Markerless Mutagenesis in Methylotuvimicrobium.

Due to their fast growth rate and robustness, some haloalkalitolerant methanotrophs from the genus Methylotuvimicrobium have recently become not only promising biocatalysts for methane conversion but also favorable materials for obtaining fundamental knowledge on methanotrophs. Here, to realize unmarked genome modification in Methylotuvimicrobium bacteria, a counterselectable marker (CSM) was developed based on pheS, which encodes the α-subunit of phenylalanyl-tRNA synthetase. Two-point mutations (T252A and A306G) were introduced into PheS in Methylotuvimicrobium buryatense 5GB1C, generating PheSAG, which can recognize p-chloro-phenylalanine (p-Cl-Phe) as a substrate. Theoretically, the expression of PheSAG in a cell will result in the incorporation of p-Cl-Phe into proteins, leading to cell death. The Ptac promoter and the ribosome-binding site region of mmoX were employed to control pheSAG, producing the pheSAG-3 CSM. M. buryatense 5GB1C harboring pheSAG-3 was extremely sensitive to 0.5 mM p-Cl-Phe. Then, a positive and counterselection cassette, PZ (only 1.5 kb in length), was constructed by combining pheSAG-3 and the zeocin resistance gene. A PZ- and PCR-based strategy was used to create the unmarked deletion of glgA1 or the whole smmo operon in M. buryatense 5GB1C and Methylotuvimicrobium alcaliphilum 20Z. The positive rates were over 92%, and the process could be accomplished in as few as eight days.

Role of the malic enzyme in metabolism of the halotolerant methanotroph Methylotuvimicrobium alcaliphilum 20Z.

The bacteria utilizing methane as a growth substrate (methanotrophs) are important constituents of the biosphere. Methanotrophs mitigate the emission of anthropogenic and natural greenhouse gas methane to the environment and are the promising agents for future biotechnologies. Many aspects of CH4 bioconversion by methanotrophs require further clarification. This study was aimed at characterizing the biochemical properties of the malic enzyme (Mae) from the halotolerant obligate methanotroph Methylotuvimicrobium alcaliphilum 20Z. The His6-tagged Mae was obtained by heterologous expression in Escherichia coli BL21 (DE3) and purified by affinity metal chelating chromatography. As determined by gel filtration and non-denaturating gradient gel electrophoresis, the molecular mass of the native enzyme is 260 kDa. The homotetrameric Mae (65x4 kDa) catalyzed an irreversible NAD+-dependent reaction of L-malate decarboxylation into pyruvate with a specific activity of 32 ± 2 units mg-1 and Km value of 5.5 ± 0.8 mM for malate and 57 ± 5 μM for NAD+. The disruption of the mae gene by insertion mutagenesis resulted in a 20-fold increase in intracellular malate level in the mutant compared to the wild type strain. Based on both enzyme and mutant properties, we conclude that the malic enzyme is involved in the control of intracellular L-malate level in Mtm. alcaliphilum 20Z. Genomic analysis has revealed that Maes present in methanotrophs fall into two different clades in the amino acid-based phylogenetic tree, but no correlation of the division with taxonomic affiliations of the host bacteria was observed.

Genome-scale evaluation of core one-carbon metabolism in gammaproteobacterial methanotrophs grown on methane and methanol

Methylotuvimicrobium alcaliphilum 20Z is a promising platform strain for bioconversion of one-carbon (C1) substrates into value-added products. To carry out robust metabolic engineering with methylotrophic bacteria and to implement C1 conversion machinery in non-native hosts, systems-level evaluation and understanding of central C1 metabolism in methanotrophs under various conditions is pivotal but yet elusive. In this study, a genome-scale integrated approach was used to provide in-depth knowledge on the metabolic pathways of M. alcaliphilum 20Z grown on methane and methanol. Systems assessment of core carbon metabolism indicated the methanol assimilation pathway is mostly coupled with the efficient Embden-Meyerhof-Parnas (EMP) pathway along with the serine cycle. In addition, an incomplete TCA cycle operated in M. alcaliphilum 20Z on methanol, which might only supply precursors for de novo synthesis but not reducing powers. Instead, it appears that the direct formaldehyde oxidation pathway supply energy for the whole metabolic system. Additionally, a comparative transcriptomic analysis in multiple gammaproteobacterial methanotrophs also revealed the transcriptional responses of central metabolism on carbon substrate change. These findings provided a systems-level understanding of carbon metabolism and new opportunities for strain design to produce relevant products from different C1-feedstocks.